We propose a collaborative effort to characterize the cellular and systems mechanisms of persistent cortical activity and its contributions to working memory in the rat. The project will leverage the advantages of trace eyelid conditioning (TEC) and combine the systems-neuroscience expertise of the Mauk lab with the cellular- neuroscience expertise of the Johnston lab. We will utilize several novel approaches designed to establish links between the cellular mechanisms of persistent activity in prefrontal cortex (PFC) and the systems mechanisms of working memory. The Mauk lab has demonstrated that TEC requires persistent activity of an interconnected pathway from medial (m) PFC to lateral pons (and then to cerebellum) that is required for TEC. The Johnston lab has injected vital retrograde tracers (Lumafluor beads) in vivo to identify in vitro a subpopulation of L5 pyramidal neurons in mPFC with unique electrophysiological properties that projects to the lateral pons. Finally, in vitro recordings from these mPFC neurons from behaviorally trained animals have been made to investigate the cellular mechanisms associated with the behavioral task. These and other findings establish TEC as an especially tractable means to study the systems and cellular mechanisms of a working- memory-related behavior. We propose 1) to use local injections of pharmacological agents and multiunit stimulation and recordings to provide a systems-level analysis of persistent firing, TEC, and working memory in the behaving rat; 2) to use local injections in vivo of vital ortho- and retrograde tracers to determine the pathways required for TEC. A 3D anatomical map of these pathways will be constructed from the data; and 3) to investigate the cellular and neuromodulatory properties of labeled neurons in these pathways that participate in the behavior. The latter experiments will be accomplished using whole-cell and cell-attached patch recordings and Ca2+ imaging from PFC neurons in brain slices. This project should permit an unprecedented analysis of persistent cortical activity and significantly enhance the prospects for treating disease-related deficits in working memory.